A Membrane‐Targeting Aggregation‐Induced Emission Probe for Monitoring Lipid Droplet Dynamics in Ischemia/Reperfusion‐Induced Cardiomyocyte Ferroptosis

Abstract Myocardial ischemia/reperfusion injury (MIRI) is the leading cause of irreversible myocardial damage. A pivotal pathogenic factor is ischemia/reperfusion (I/R)‐induced cardiomyocyte ferroptosis, marked by iron overload and lipid peroxidation. However, the impact of lipid droplet (LD) changes on I/R‐induced cardiomyocyte ferroptosis is unclear. In this study, an aggregation‐induced emission probe, TPABTBP is developed that is used for imaging dynamic changes in LD during myocardial I/R‐induced ferroptosis. TPABTBP exhibits excellent LD‐specificity, superior capability for monitoring lipophagy, and remarkable photostability. Molecular dynamics (MD) simulation and super‐resolution fluorescence imaging demonstrate that the TPABTBP is specifically localized to the phospholipid monolayer membrane of LDs. Imaging LDs in cardiomyocytes and myocardial tissue in model mice with MIRI reveals that the LD accumulation level increase in the early reperfusion stage (0–9 h) but decrease in the late reperfusion stage (>24 h) via lipophagy. The inhibition of LD breakdown significantly reduces the lipid peroxidation level in cardiomyocytes. Furthermore, it is demonstrated that chloroquine (CQ), an FDA‐approved autophagy modulator, can inhibit ferroptosis, thereby attenuating MIRI in mice. This study describes the dynamic changes in LD during myocardial ischemia injury and suggests a potential therapeutic target for early MIRI intervention.


Introduction
Acute myocardial infarction remains a predominant cause of global mortality and morbidity. [1]The principal approach to limit infarct size and subsequent ventricular remodeling involves reperfusion therapy, typically achieved through primary percutaneous coronary intervention (PCI) or thrombolysis. [2]However, reperfusion can provoke irreversible damage to cardiomyocytes, a phenomenon recognized as myocardial ischemia/reperfusion injury (MIRI). [3]Despite advancements in PCI techniques and the availability of antiplatelet and antithrombotic medications, there are currently no established treatments that conclusively mitigate MIRI.There is an urgent need for the development of novel cardioprotective strategies aimed at mitigating MIRI and improving clinical outcomes in AMI patients.
Ferroptosis, characterized by irondependent lipid peroxidation, has emerged as a novel form of regulated cell death. [4]ccumulating evidence indicates that ferroptosis is the primary modality of cardiomyocyte death in MIRI. [5]Fang et al. demonstrated for the first time that during the ischemia/reperfusion (I/R) period, the degradation of ferritin releases iron, triggering the iron-mediated Fenton reaction in mitochondria, which leads to lipid peroxidation and subsequent cardiac injury. [6]Subsequent studies by Hiroyuki Tsutsui et al. reveal that hypoxia/reoxygenation augments Heme oxygenase 1 level, inducing iron overload in the endoplasmic reticulum, thereby fostering excessive lipid peroxide generation and subsequent ferroptosis in cardiomyocytes. [7]The inhibition of lipid peroxidation represents a promising strategy for mitigating ferroptosis and enhancing cardiac function in MIRI. [8]Lipid droplets (LDs) act as modulators of lipid peroxidation, and their degradation increases free fatty acid (FFA) production and promotes ferroptosis. [9]Nevertheless, the precise alterations in LD metabolism associated with ferroptosis following MIRI remain incompletely understood.Hence, visualizing LD dynamics assumes critical importance not only in elucidating the interplay between LDs and ferroptosis progression but also in fostering the development of novel ferroptosis inhibitors for MIRI treatment.
Fluorescence imaging (FI) using small-molecule probes has emerged as a valuable technique for visualizing biomolecules at subcellular levels, particularly in vivo, owing to its noninvasiveness, real-time, excellent spatial resolution, and high signal-to-noise ratio (SNR).Although commercial probes like Nile Red and BODIPY493/503 are widely utilized for LD staining, they often exhibit non-specific staining of other hydrophobic cellular structures, thereby compromising the signal-to-noise ratio.Recently, numerous fluorescent probes tailored specifically for LD imaging have been developed (Table S1, Supporting Information).However, these probes have certain limitations in their photophysical properties, which impede their potential applications as sensitive and specific fluorescent probes.Most of these fluorescent probes consist of highly hydrophobic conjugated aromatic groups, which tend to aggregate in an intracellular milieu, leading to an aggregation-induced quenching (ACQ) effect on the fluorescence signal. [10]Consequently, they are typically used at low concentrations due to the ACQ effect, resulting in low photo-bleaching resistance. [11]Furthermore, the majority of LD-specific probes exhibit a small Stokes shift (<100 nm), which may compromise image quality in confocal microscopy measurements. [12]Given their remarkable brightness and high photostability, fluorescent probes featuring aggregation-induced emission (AIE) characteristics have attracted considerable attention for bioimaging applications. [13]Recently, several AIE-based fluorescent probes have been developed for LD imaging at both cellular and tissue levels.These probes not only enable LD imaging but also facilitate the differentiation of cancer cells and the visualization of LDs in tumor tissues, liver tissues, or atherosclerotic plaque. [14]However, there remains a significant gap in understanding the in vivo dynamics and accumulation of LDs during disease progression and treatment.Specifically, there is a lack of in vivo investigation into dynamic LD changes during MIRI.Therefore, it is imperative to employ AIE probes for specific LD recognition and integrate this approach with MIRI models to elucidate the correlation between ischemia/reperfusion-induced cardiomyocyte ferroptosis and LD abnormalities.
Herein, we have developed LD-targeting molecular probes (TPABTBP, PMBTDP, and BTDPP) by combining intramolecular charge transfer (ICT) with AIE.Among these probes, TPABTBP showed polarity-sensitive fluorescence emission and excellent performance for specifically imaging LDs in live cells.We reveal for the first time that the probe TPABTBP is trapped in the phospholipid LD monolayer.Facilitated by TPABTBP, we revealed that the LD accumulation level increased in the early reperfusion stage but decreased in the late reperfusion stage via lipophagy.The degradation of LDs promoted lipid peroxidation, exacerbating the myocardial injury.Importantly, we demonstrate that chloroquine (CQ) inhibits LD breakdown and ameliorates MIRI in murine models (Scheme 1).

Design and Synthesis of AIE-based Probes for Imaging LDs
LDs consist of a neutral lipid core containing triacylglycerols (TAGs) and steryl esters, encased within a phospholipid monolayer adorned with specific proteins on its surface.This inherent high hydrophobicity makes LDs an ideal target for imaging. [15]raditionally, fluorescent probes designed for LD imaging utilize highly lipophilic molecular scaffolds with intramolecular charge transfer (ICT) characteristics.However, such probes often exhibit fluorescence in polar media, resulting in significant background emission.To address this challenge, we developed LDtargeted probes with both ICT and AIE properties based on the following considerations.ICT-based probes demonstrate a blue shift in emission wavelength and increased fluorescence intensity in nonpolar environments, making them ideal for LD imaging with minimal background interference.Additionally, ICTbased probes offer substantial Stokes shifts, contrasting with the limited Stokes shifts observed in commercial LD probes like Nile Red and BODIPY 493/503, which enhances LD image quality by reducing incident light interference.Conventional dyes are susceptible to aggregation-caused quenching (ACQ), leading to poor resistance to photo-bleaching.In contrast, AIEgens aggregate within cellular LDs, allowing them to emit fluorescence and exhibit excellent photostability (Figure 1a).The molecular structure of the fluorescent probe, illustrated in Figure S1 (Supporting Information), comprises tetra-aryl imidazolyl or triphenylamine as the electron donor (D) and benzothiadiazole and pyridine as the acceptor (A), forming a typical D-A molecular structure.The rotation of the electron donor (D) tetra-aryl imidazolyl and acceptor (A) triphenylamine imparts the molecule with both AIE and ICT properties.

Characteristics of the Photophysical Properties
Molecules featuring a donor (D)--acceptor (A) configuration exhibit a significant solvatochromic effect, as evidenced by changes in their photophysical properties in response to variations in solvent polarity.Therefore, we analyzed the photoluminescence (PL) spectra of TPABTBP, PMBTDP, and BTDPP in solvents with different polarities.These compounds exhibited high-intensity fluorescence in a weak polar solvent, toluene, with emission wavelengths of 600, 575, and 580 nm, respectively (Figure 1b-d).The fluorescence quantum yields of TPABTBP, PMBTDP, and BT-DPP in toluene were 50.9%, 99.97%, and 98.95%, respectively (Table S2, Supporting Information).
In toluene, TPABTBP showed a red shift of emissions from 600 to 645 nm in methanol, accompanied by a sharp decrease in fluorescence intensity.With increasing solvent polarity, the fluorescence intensity of PMBTDP and BTDPP decreased markedly, accompanied by a redshift in the emission wavelength.These solvent polarity-dependent fluorescence emission properties were attributed to the ICT effect contributed by the donation of elec-trons (methoxy-substituted triphenylamine, tetra-aryl imidazole, and triphenylamine) to an electron-withdrawing group in the benzothiadiazole core.ICT molecules display distinct fluorescence responses in polar and nonpolar solvents.In polar solvents, they experience a more significant relaxation effect, causing a bathochromic-shifted fluorescence with reduced quantum yield.In contrast, nonpolar solvents induce less ICT effect, leading to emissions at shorter wavelengths with increased fluorescence intensity. [16]Furthermore, the ICT effect of TPABTBP, PMBTDP, and BTDPP was confirmed by density functional theory calculations (Figure 1e-g).The lowest unoccupied molecular orbital (LUMO) of TPABTBP, PMBTDP, and BTDPP were predominately occupied on the strong electron-accepting group of the benzothiadiazole core, while the highest occupied molecular orbital (HOMO) was mainly concentrated in electron-donating group (methoxy-substituted triphenylamine, tetra-aryl imidazole, and triphenylamine).14c,17] The calculated band gaps (△E) of PMBTDP, BTDPP, and TPABTBP were 2.87, 2.82, and 2.52 eV, respectively, suggesting a gradual enhancement of the ICT effect.LD polarity has been shown to be close to that of toluene. [18]The fluorescence responses of the compounds TPABTBP and PMBTDP in toluene solution indicated that they were suitable for LD imaging.
In addition to solvatochromism, TPABTBP, PMBTDP, and BT-DPP showed AIE.In DMSO, they also showed weak fluorescence emission in DMSO solution.TPABTBP and PMBTDP showed fluorescence quantum yields below 0.1% in DMSO, while BT-DPP demonstrated a fluorescence quantum yield of 2.08% in DMSO (Table S2, Supporting Information).When the water content was increased to 80%, the fluorescence emission intensity of TPABTBP, PMBTDP, and BTDPP, reached the maximum level (Figure 1h-m).In aqueous solutions, TPABTBP, PMBTDP, and BTDPP tended to aggregate due to their poor solubility.This aggregation limited their intramolecular motion and triggered AIE.Unexpectedly, we observed a reduction in emission intensity and a slight redshift in the emission spectra of TPABTBP, PMBTDP, and BTDPP when the water content was increased from 80% to 95%.The fluorescence quantum yields of TPABTBP, PMBTDP, and BTDPP in H 2 O (containing 5% DMSO) were 6.53%, 12.86%, and 53.28%, respectively (Table S2, Supporting Information).These results can potentially be explained by two factors: first, the crystallization-induced emission characteristics of TPABTBP, PMBTDP, and BTDPP [19] and second, the effect of aggregate size. [20]TPABTBP, PMBTDP, and BTDPP may undergo crystalline aggregates in low water fractions.However, when the water fraction exceeds 80%, these molecules tend to rapidly aggregate into smaller sizes with reduced emissive. [21]The emission of small-sized aggregates may be more susceptible to the influence of the surrounding solvent environment, resulting in a reduction in emission intensity.
To verify the presence of nanoaggregates in the DMSO/water, dynamic light scattering (DLS) experiments were performed.The DLS data shown in Figure S12 (Supporting Information) revealed that the size of the TPABTBP aggregates decreased from ≈196.3 nm at an 80 vol% water fraction to ≈146.4 nm at a 95 vol% water fraction.Similarly, when the water content in the system increased from 80% to 95%, the sizes of the PMBTDP and BTDPP aggregates reduced from 174.0 and 181.6 nm to 116.5 and 159.3 nm, respectively.We further measured the UV absorption spectra of TPABTBP, PMBTDP, and BTDPP in aqueous solutions (containing 5% DMSO).As shown in Figure S13 (Supporting Information), significant tailing phenomena were observed in the UV absorption spectra of TPABTBP, PMBTDP, and BTDPP.Specifically, TPABTBP and BTDPP showed distinct absorption peaks at 470 and 480 nm, respectively.The tailing phenomenon in the UV absorption spectra, induced by Mie scattering, further confirmed the formation of aggregates of TPABTBP, PMBTDP, and BTDPP in aqueous solutions.This observation is consistent with the results obtained from Dynamic Light Scattering (DLS) measurements.The results above indicate that in the mixed system of DMSO and water when the water content reaches 80%, the probes TPABTBP, PMBTDP, and BTDPP form aggregates, restricting intramolecular rotation and generating fluorescence.When the water content exceeds 80%, the decrease in fluorescence could possibly be attributed to a reduction in aggregate size.Generally, the larger the aggregate size, the brighter it is. [22]Their polarity-responsive and AIE properties make TPABTBP, PMBTDP, and BTDPP promising probes for LD imaging.

Specific Fluorescent Imaging of LDs in Live Cells
To verify the capacity of the probes (PMBTDP, BTDPP, and TPABTBP) for LD imaging in live cells, the colocalization experiments were performed using BODIPY 493/503, a commercial LD dye.As shown in Figure S14a-c (Supporting Information), the bright red fluorescence signals emitted by PMBTDP, BTDPP, and TPABTBP overlapped precisely with the green emission sig-nals from BODIPY 493/503.Pearson's correlation coefficients were 0.93, 0.87, and 0.96 for PMBTDP, BTDPP, and TPABTBP, respectively, indicating that PMBTDP, BTDPP, and TPABTBP specifically targeted and stained LDs in living cells.The ClogPs of PMBTDP (ClogP = 8.65), BTDPP (ClogP = 7.21), and TPABTBP (ClogP = 7.16) were calculated using XlogP3 [23] and were significantly higher than the ClogP of BODIPY 493/503 (ClogP = 2.98).Because the LDs are less polar than certain other lipid structures, probes with higher ClogP values are better suited for LD imaging.Therefore, the appropriate lipophilic properties of PMBTDP, BTDPP, and TPABTBP contributed to their ability to target LD.Because TPABTBP showed weak fluorescence in polar solvents but strongly in nonpolar systems with emission wavelengths exceeding 600 nm, we selected TPABTBP for further research.To explore distractors in the organism and the effect of different pH on TPABTBP, we conducted an in vitro assessment of the interference caused by biomolecules (HSA, ATP, ADP, GSH, Cys, glucose and RNA), metal ions (Na + , K + , Fe 2+ , Fe 3+ , Ca 2+ ), and pH on the fluorescence emission of the TPABTBP.As shown in Figure S15a,b (Supporting Information), the fluorescence intensity of TPABTBP mixed with various potential interferents did not exhibit significant changes.This observation indicates that TPABTBP possesses excellent anti-interference capability in complex biological systems, making it advantageous for the specific imaging of LDs.
To determine the appropriate concentration of TPABTBP for LD imaging, HepG2 cells were subjected to bright-field and fluorescence microscopy observations to evaluate the staining of LDs.Due to its higher refractive index, LDs are prominently visible as dark spots in phase-contrast microscopy images.As shown in Figure S16 (Supporting Information), at a concentration of 5 μM, only a limited number of brightly fluorescent droplets, stained with TPABTBP, demonstrated a well-pronounced merge with distinct black dots.In contrast, when the concentration of TPABTBP was increased to 30 μM, nearly all LDs in the cytoplasm were effectively stained with TPABTBP.Although 40 and 50 μM TPABTBP still provide good imaging results for LDs, considering the potential cytotoxicity caused by high concentrations of the probe and cost issues, we ultimately selected 30 μM as the final imaging concentration.Subsequently, we investigated whether concentration affects the co-localization of TPABTBP with LDs.As demonstrated in Figure S17 (Supporting Information), after incubation of HeLa cells with various concentrations (5, 10, 30, 40, and 50 μM) of TPABTBP, robust fluorescent signals were detected in the red emission channel, and these signals closely overlapped those emitted by BODIPY 493/503.The Pearson's coefficients were 0.90, 0.90, 0.94, 0.91 and 0.88, respectively.Investigations into higher concentrations revealed no significant improvement in imaging quality.Based on the above results, we believe that 30 μM is the appropriate concentration for imaging LDs using TPABTBP.Additionally, we investigated the LD targeting efficiency of TPABTBP in various living cells, including H9c2 and RAW264.7 cells (Figure S18a,b, Supporting Information).Science the colocalization coefficients of TPABTBP and BODIPY 493/503 were 0.92, and their fluorescence signals closely overlapped, we concluded that TPABTBP effectively images LDs across different cell types.
To further confirm the specificity of TPABTBP for LD imaging, immunofluorescence colocalization analysis of the PLIN2 protein, which is highly expressed on the LD membrane, was conducted. [24]As shown in Figure S19 (Supporting Information), the red fluorescence signal (indicating LD staining) overlapped well with the green fluorescence signal (representing PLIN2), demonstrating the superior specificity of TPABTBP for LD imaging.Additionally, the co-localization of TPABTBP with other organelles was evaluated to confirm its labeling specificity for LDs.As depicted in Figure 2, TPABTBP showed minimal colocalization with commercial probes for mitochondrial (Mito), endoplasmic reticulum (ER), Golgi apparatus (GA), and lysosomal (Lyso).The Pearson coefficients between TPABTBP and Mito, ER, GA, and Lyso were −0.11, −0.07, 0.02, and 0.04, respectively.The morphology of LDs visualized by TPABTBP was distinctly clear and appeared nearly spherical.The red fluorescence signal generated by TPABTBP displayed excellent colocalization with the green fluorescence signal of BODIPY 493/503, with a high Pearson correlation coefficient of 0.9.These results collectively demonstrate the excellent specificity of TPABTBP for LD imaging.

Validating the Advantage of TPABTBP Relative to BODIPY493/503
Compared to the widely used BODIPY 493/503, TPABTBP offers three distinct advantages.Firstly, TPABTBP exhibits higher specificity for LD staining compared to BODIPY 493/503.We evaluated whether TPABTBP and BODIPY 493/503 exhibited non-specific staining of lysosomes during imaging.As shown in Figure S20 (Supporting Information), there was a substantial overlap between the fluorescence signals of BODIPY 493/503 and Lyso-Tracker, indicating potential non-specific staining of lysosomes by BODIPY 493/503, consistent with previous findings. [25]In contrast, the fluorescence signal of TPABTBP remained distinctly separate from the signal of lysosomes, confirming its excellent LD selectivity.Additionally, owing to its AIE characteristics, TPABTBP exhibits superior photostability compared to BODIPY 493/503.A photobleaching experiment conducted on HepG2 cells co-incubated with BODIPY 493/503 and TPABTBP for 30 min revealed notable differences.As depicted in Figure S21a,b (Supporting Information), while the green fluorescence of BODIPY 493/503 rapidly decayed by 70% within 20 s under the same laser excitation power, TPABTBP only exhibited a 6% decrease.Even after continuous excitation for 240 s, the fluorescence intensity of BODIPY 493/503 decreased by ≈90%, whereas TPABTBP showed only ≈30% reduction, confirming its superior anti-photobleaching properties.Moreover, the signal-tonoise ratio (SNR) of TPABTBP imaging of LDs surpassed that of BODIPY 493/503.SNR calculations conducted for both probes further corroborated their imaging performance.As shown in Figure S22a (Supporting Information), the red fluorescence signals of TPABTBP appeared clear and bright, while the green fluorescence signal from BODIPY 493/503 appeared diffuse and blurry throughout the cell.Analysis of ten randomly selected regions for SNR calculation indicated that the SNR of TPABTBP was 2.2 times higher than that of BODIPY 493/503 (Figure S22b, Supporting Information).This establishes a foundation for the application of TPABTBP to explore the dynamics of LDs.These results demonstrate that compared to the commercial LD probe BODIPY 493/503, TPABTBP possesses superior LD imaging capabilities.

Molecular Dynamics (MD) Simulation of the Interaction Between the Probe and LDs
To assess TPABTBP's ability to penetrate the phospholipid monolayer membrane of LDs, we conducted all-atom molecular dynamics (MD) simulations to track TPABTBP migration from the cytosol to the LD core.The construction of the simulated LD system involved three primary steps: 1) constructing the phospholipid membrane system; 2) creating the triacylglycerol (TAG) slab; and 3) inserting the TAG layer between the upper and lower phospholipid monolayers to form the complete simulation system.
Firstly, a solvated phospholipid bilayer (≈ 2 nm thick) was constructed and equilibrated in an isothermal-isobaric (NPT) ensemble for 100 ns.As depicted in Figure S23 (Supporting Information), after 100 ns of equilibration, the phospholipid membrane system exhibited convergence in terms of the surface area per lipid molecule (APL), phospholipid density, energy of the phospholipid membrane system, and temperature of the simulation system.This convergence indicated that the phospholipid membrane had reached an equilibrium state.Secondly, we used Packmol software to build TAG Slab (10 nm-thick).Prior to assembling the complete simulation system, a separate 20 ns NPT simulation was conducted on the TAG slab under conditions of 300 K and 1 bar to attain an equilibrium state.After 20 ns of equilibration, the energy of the system and the density of TAG within the TAG slab stabilized, indicating convergence (Figure S24, Supporting Information).Finally, we inserted the equilibrated TAG slab between the upper and lower phospholipid monolayers and conducted a 10 ns NPT equilibration simulation of the combined system.After 10 ns of equilibrium simulation, the system's density, energy, and surface area per lipid molecule exhibited stabilization, indicating that the LD simulation system had reached a stable and converged state (Figure S25, Supporting Information).
Our LD model comprised two interfaces: one at the interface between the cytosol and phospholipid monolayer (interface I), and the other at the boundary between the phospho-lipid monolayer and TAGs (interface II).Through unrestricted MD simulations and meta-dynamics calculations, we investigated TPABTBP's migration process across these boundaries.At 100 ns, TPABTBP began to cross interface I and had completely entered the phospholipid monolayer by 200 ns (Figure 3a).However, TPABTBP was trapped in the phospholipid monolayer during the 200-500 ns simulation and failed to pass interface II.We quantified TPABTBP's end-to-end distance (R end-to-end ) to investigate its structural changes during LD migration, revealing a constant probe length of ≈1.3 nm, indicating rigidity (Figure 3b).The migration depth of TPABTBP relative to the phospholipid monolayer showed rapid infiltration upon reaching interface I, with subsequent stalling within the monolayer during the 500-ns stimulation (Figure 3c).Owing to the rigid structural characteristics of TPABTBP, the variation conformational order parameter (μ) was independent of the R end-to-end and solely reflected alteration changes (Figure S26, Supporting Information).To better assess TPABTBP's orientation, we measured the angle between the R end-to-end vector and the membrane normal (Figure S27, Supporting Information).The rigid structure of TPABTBP likely creates a substantial free energy barrier during penetration into the LD core, as geometric rearrangements are required to accommodate the unfolded probe within interlaced TAG chains.Multi-walker well-tempered meta-dynamics simulations with four replica walkers were conducted to obtain the free energy landscape when TPABTBP crossed the two interfaces. [26]he separate replicas are shown in Figures S28 and S29 (Supporting Information).As shown in Figure 3d, TPABTBP exhibited the lowest free energy (−63.36 ± 3.75 kJ mol −1 ) at a maximum migration depth of −1.52 ± 0.05 nm, corresponding to an angle of 2.04 ± 0.08 radians between the R end-to-end vector and the membrane normal.This finding confirmed that TPABTBP cannot migrate to the core of LDs, localizing instead to the phospholipid monolayer membrane.The MD simulation revealed that TPABTBP localized to the monolayer phospholipid membrane structure of LDs.The low-polarity environment of the phospholipid monolayer promotes the fluorescence emission of TPABTBP.Additionally, the parallel arrangement of long phospholipid chains in the monolayer helps restrict the intramolecular rotation of TPABTBP, further enhancing fluorescence emission.Consistent with previous findings, our results suggested that rigid lipid molecules such as TPABTBP cannot cross the LD phospholipid monolayer membrane to enter the lipid core. [27]

Experimentally Validate that TPABTBP Specifically Localized to the Phospholipid Monolayer Membrane of LDs
To validate the specific targeting of TPABTBP to the lipid monolayers on LDs, we conducted a series of experiments employing TPABTBP for imaging LDs.Initially, LDs were extracted from HepG2 cells utilizing an LD isolation kit and then incubated with TPABTBP at room temperature for 3 min prior to microscopic observation.In Figure S30 (Supporting Information), LDs are characterized as red hollow circular structures.
Further examination involved magnifying LDs with a 100x oil objective during laser confocal microscopy imaging of HepG2 cells, where TPABTBP appeared as red rings surrounding the LDs, as illustrated in Figure S31 (Supporting Information).This pattern was consistently observed in H9c2 cells under identical magnification, reinforcing the membrane-targeting capabilities of TPABTBP.To capture the dynamics of this process, insitu time-lapse confocal imaging was employed, with Video S1 (Supporting Information) demonstrating the distinctive red, hollow, ring-shaped patterns of LDs in the cytoplasm, confirming TPABTBP's specific targeting.Moreover, a HepG2 cell line engineered to express GFP-tagged PLIN2, [28] a protein associated with the LD membrane, was used.Following oleic acid (OA) treatment to induce LD formation, these cells were stained with TPABTBP and analyzed under 1000x magnification using fluorescence microscopy.Figure S32 (Supporting Information) shows the colocalization of GFP-PLIN2's green fluorescence with TPABTBP's red fluorescence, particularly notable at higher magnifications, suggesting a specific association of TPABTBP with the LD membrane.
Further insights were gained through structured illumination microscopy (SIM).H9c2 cells were incubated with 30 μM TPABTBP for 30 min to stain LDs.As shown in Figure 4a, SIM images showed the distribution of spherical LDs in the cytoplasm.By increasing the magnification of the image, the ring-shaped red fluorescence pattern of the LDs is clearly seen, indicating that TPABTBP was localized mainly on the LD monolayer membrane (Figure 4b,c).Due to the excellent spatial resolution of SIM imaging, the diffraction limit of TPABTBP in imaging LDs reached 61 nm (Figure 4d).We also observed similar hollow rings in other LDs (Figure 4e,f).Especially, the spectral analysis of the selected LD signals revealed a decrease in the red fluorescence signal of TPABTBP at the core of the LDs, while it was enhanced at the LD membrane.These results indicate the binding of TPABTBP to the monolayer phospholipid mem-brane of LDs, and it was consistent with the MD simulations.We further assessed the target specificity of TPABTBP toward LD membranes using cells with large LDs, such as HepG2 cells known for their active lipid metabolism. [29]As shown in Figure 4g-i, LDs exhibited a distinctive hollow ring-like structure.Notably, after a layer-by-layer scan of LD structures using SIM, 3D structural fitting images unequivocally demonstrated that, from a different rotational perspective, TPABTBP signals were localized on the LD membrane (Figure 4j-l).These findings provide further confirmation of TPABTBP's specific targeting of the monolayer phospholipid membrane of LDs.

Imaging the Dynamic Changes in LDs During Ferroptosis
Recent studies have highlighted a close relationship between myocardial ischemia-reperfusion injury (MIRI) and ferroptosis. [30]lthough ferroptosis is driven by lipid peroxidation, [5,31] the impact of LDs on the dynamic progression of ferroptosis remains unclear.To investigate ferroptosis, we treated H9c2 and RAW264.7 cells with Erastin, a well-known inducer of ferroptosis, for 24 h, followed by co-staining with TPABTBP and BODIPY 493/503.In the absence of Erastin, LDs were observed as punctate structures distributed throughout the cytoplasm of both RAW 264.7 and H9c2 cells.However, upon Erastin treatment (5 μM), there was a significant increase in both the number and mean diameter of LDs in both cell types (Figure 5a,b).To provide quantitative evidence for this phenomenon, we quantified TPABTBP fluorescence intensity in RAW 264.7 and H9c2 cells.In comparison to the control group, after induction for 24 h, the mean fluorescence intensity (MFI) in RAW264.7 and H9c2 cells were 2.0 and 2.8 times higher, respectively (Figure S33a,b, Supporting Information).The increase in the number of LDs during ferroptosis indicated that LDs may have antioxidative functions, and they store polyunsaturated fatty acids to protect cells from lipotoxicity and oxidative stress. [32]o mimic MIRI in vitro, H9c2 cells were subjected to induce hypoxia/reoxygenation (H/R) injury. [33]C11-BODIPY 581/591 was utilized to assess the extent of lipid peroxidation in H9c2 cells during the H/R process.Lipid peroxidation results in a transition of the fluorescence emission peak of C11-BODIPY 581/591 from red light (≈ 590 nm) in the reduced state to green light (≈510 nm) in the oxidized state.H9c2 cells in the normal group exhibited strong red fluorescence with weak green fluorescence, indicating low levels of lipid peroxidation (Figure S34, Supporting Information; Figure 5c).However, after 12 h of hypoxia followed by 2 h of reoxygenation, the MFI of the green channel increased significantly, while the MFI of the red channel decreased markedly by 75.97%, indicating a notable increase in lipid peroxidation (Figure S34c, Supporting Information; Figure 5d).Flow cytometry analysis showed that the lipid peroxidation rate of cardiomyocytes in the H/R treatment group was 5.7-fold (21.58% vs. 3.77%) higher than that of the normoxia group (Figure 5e).Subsequently, we have performed additional experiments using the Calcein-AM/PI cytotoxicity assay kit to evaluate cell death under conditions of hypoxia-reoxygenation (H/R) and Erastin induction.The Calcein-AM/PI assay enables the discrimination between live and dead cells, with Calcein-AM emitting intense green fluorescence in live cells, and PI labeling dead cells with red fluorescence.In Figure S35a (Supporting Information), the red fluorescence signal of H9c2 cells significantly increased after 12 h of hypoxia followed by 2 h of reoxygenation compared to the Normal group, indicating an elevated number of dead cells.The quantitative results of live cells labeled with Calcein-AM indicated that the proportion of live cells in the H/R group decreased to 27.4% (Figure S35b, Supporting Information).These results indicated that simulating ferroptosis in vitro through hypoxiareoxygenation indeed leads to a substantial increase in cell death.
Additionally, we used the probe TPABTBP to explore how LDs in cardiomyocytes respond to H/R treatment.As illustrated in Figure 5f,g, the number of LDs was significantly increased after H/R treatment.This change aligned with the changes in RAW 264.7 and H9c2 cells when Erastin was used to induce ferroptosis.A quantitative flow cytometry analysis showed that LD content in H9c2 cells after H/R treatment was 5.8-fold that of the normoxia group (Figure 5h).These findings reveal that H/R treatment induces ferroptosis in living cells, and this process was accompanied by a marked increase in the number of LDs in the cytoplasm.
To explore LD dynamics during ferroptosis, we induced Erastin in HepG2, H9c2, and Hela cells, and then monitored LD changes at various time points through imaging.As depicted in Figure S36a,b (Supporting Information), the quantity of LDs increased during the early stages of ferroptosis induction (0-9 h), followed by a decline during the later stages (12-24 h).In order to reveal the correlation between the trends in LDs and intracellular lipid peroxidation levels, we further investigated the time-dependent changes in LDs and lipid peroxidation during ferroptosis.H9c2 cells were treated with 5 μM Erastin at various time points (0, 3, 6, 9, 12, and 24 h), labeled with Oil Red O, and treated with C11-BODIPY 581/591 and TPABTBP.The results indicated that with the prolonged induction time of Erastin, the intracellular lipid peroxidation levels continued to increase (Figure 5i; Figure S37, Supporting Information), while LDs exhibited a trend of initially increasing (0-9 h) and then decreasing (Figure 5j; Figure S38, Supporting Information).Furthermore, Erastin-induced ferroptosis in H9c2 cells was monitored for changes in cell death using Calcein-AM/PI staining.As depicted in Figure S39a (Supporting Information), the PI fluorescence signal within the cells exhibited a progressive enhancement after 3 h of Erastin induction, reaching a point at 24 h where the majority of cells were labeled with red PI fluorescence.Meanwhile, the Calcein-AM fluorescence signal, indicative of live cells, remained exceedingly faint.These observations suggested a notable escalation in the cell death rate with increasing duration of Erastin exposure.Moreover, the proportion of viable cells was quantified by assessing the variations in Calcein-AM fluorescence intensity.The results revealed that after 24 h of Erastin induction, the ratio of live H9c2 cells significantly decreased to only 8.46% (Figure S39b, Supporting Information).Thus, these findings demonstrated that cell death is specific to hypoxia-reoxygenation (H/R) and Erastin-induced ferroptosis.In addition, we used flow cytometry to quantify the number of LDs and the level of lipid peroxidation.As depicted in Figure S40 (Supporting Information), the number of LDs continuously increased from 0 to 9 h, peaking at 9 h when the number of LDs was 5.9 times that of the 0-h group.However, as the induction time with Erastin increased to 12 h, the number of LDs began to decrease.The timing of the changes in LD number in the cytoplasm was completely different from that of the changes in lipid peroxidation level.The level of lipid peroxidation further increased with increasing Erastin treatment time, indicating exacerbated ferroptosis.These results indicated that LD synthesis increases in the early stage, but LDs begin to breakdown in the late stage of ferroptosis.
LDs are not static organelles but increase/decrease in numbers influenced by a long list of biological stimuli such as hormones, energy substrate availability, and hypoxia/reperfusion.Therefore, we used H9c2 and HepG2 cells with different levels of LDs to test the involvement of LDs in Erastin-induced ferroptosis.As a monounsaturated fatty acid, OA was widely used to promote LD synthesis in living cells. [34]Therefore, H9c2 and HepG2 cells were pre-treated with 100 μM OA for 16 h.Untreated H9c2 and HepG2 cells were used as controls.Subsequently, we assessed LD content and the degree of lipid peroxidation by using TPABTBP and C11-BODIPY during Erastin-induced ferroptosis.
After OA treatment, both H9c2 and HepG2 cells exhibited a significant increase in LD content (Figure S41a,e, Supporting Information).Specifically, LD content increased ≈2.3-fold in H9c2 cells and 3.1-fold in HepG2 cells compared to untreated cells (Figure S41b,f, Supporting Information).However, the levels of lipid peroxidation in both cell lines did not show a significant difference with or without OA pretreatment (Figure S41d,h, Supporting Information).After 9 h of Erastin induction, there was a significant increase in LD content in both H9c2 and HepG2 cells (Figure S41a,e, Supporting Information).In H9c2 and HepG2 cells without OA pretreatment, the LD content increased by 9.6 and 15.9-fold, respectively (Figure S41b,f, Supporting Information).In OA-pre-treated H9c2 and HepG2 cells, the LD content increased by 9.8 and 7.2-fold, respectively (Figure S41b,f, Supporting Information).After 9 h of Erastin induction, lipid peroxidation levels increased in H9c2 and HepG2 cells but remained at relatively lower levels (Figure S41c,g, Supporting Information).However, compared to the 9 h time point, after 20 h of Erastin induction, the LD content decreased by 77.35% and 86.96% in H9c2 and HepG2 cells without OA pretreatment (Figure S41b,f, Supporting Information), while lipid peroxidation levels increased by 3.9 and 2.9-fold, respectively (Figure S41d,h, Supporting Information).Similarly, in OA-pretreated H9c2 and HepG2 cells, the LD content decreased by 68.72% and 78.27%, respectively (Figure S41b,f, Supporting Information), and the lipid peroxidation levels increased by 3.4-and 2.2-fold, respectively (Figure S41d,h, Supporting Information).Interestingly, after 20 h of Erastin induction, cells pretreated with OA exhibited lower levels of lipid peroxidation compared to untreated cells.34a,35] These results suggest that during ferroptosis, there is an initial increase in cellular LDs followed by a subsequent decrease, irrespective of the initial LD content.In the early stages, cells synthesize new LDs to store free fatty acids as a defense mechanism against lipid peroxidation. [31a] However, in the later stages, the breakdown of LDs may release fatty acids, promoting cellular ferroptosis. [36]

LDs are Degraded via the Lipophagy Pathway, and Inhibiting LD Breakdown Alleviates Ferroptosis
9a,37] To visualize this process, we utilized transmission electron microscopy (TEM) to examine variations in LD morphology and structure in H9c2 cells before and after Erastin treatment.As shown in Figure 6a, in the absence of Erastin, the LDs in H9c2 cells exhibited a regular shape and smooth surface, with an average size of 0.4 μm.However, after treatment with Erastin (5 μM) for 24 h, the number of LDs in the cytoplasm increased, exhibiting irregular shapes and sizes.Notably, many LDs were closely associated with autophagic vesicles, indicating the involvement of autophagy in LD breakdown during ferroptosis (Figure 6b).
To validate the essential role of the autophagic machinery in these processes, we examined the autophagic flux following induction with 3-methyladenine (3-MA) or rapamycin (RAPA) during Erastin-induced ferroptosis in H9c2 cells.The mCherry-eGFP-LC3 adenovirus was employed for monitoring alterations at various phases of the autophagy process.When the autophagosome fuses with lysosome, the green fluorescence signal originating from eGFP dissipates owing to the sensitivity of eGFP to acidic conditions. [38]Thus, the yellow-fluorescent and redfluorescent puncta represent the formation of autophagosomes and autolysosomes, respectively (Figure S42a, Supporting Information).As shown in Figure S42b,c (Supporting Information), distinct red fluorescence puncta (autolysosomes) were clearly visible, indicating the occurrence of autophagy accompanying Erastin-induced ferroptosis.In the Erastin/3-MA group, both the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) decreased, indicating suppressed autophagic flux (Figure S42d,e, Supporting Information).This is attributed to 3-MA blocking class III PI-3K and inhibiting autophagophore formation. [39]Conversely, in the Erastin/RAPA group, the number of autolysosomes significantly increased (Figure S42f,g, Supporting Information), reflecting unimpeded autophagy due to RAPA inhibiting the mTOR pathway and enhancing autophagosome formation. [38]Overall, these findings indicated that 3-MA inhibits autophagic flux, while RAPA promotes it during Erastin-induced ferroptosis.In addition, we generated ATG5 gene-deficient cells to provide additional confirmation of the essential role of autophagy in LD breakdown.The selection of ATG5 gene for knockout was based on its pivotal function in orchestrating the elongation of cell membranes within autophagosomes, establishing its significance as a key autophagy-related gene. [40]irstly, the successful deletion of the ATG5 gene was confirmed through PCR and Sanger sequencing (Tables S3 and S4 and Figure S43, Supporting Information).Subsequently, we assessed the autophagy flux in ATG5 KO H9c2 cells, which are unable to form autophagophores.As shown in Figure S44 (Supporting Information), we observed an increase in green fluorescence signal in ATG5 KO cells during Erastin induction compared to wild-type (WT) H9c2 cells.Additionally, the fluorescent puncta content of autolysosomes in ATG5 KO cells exhibited a notably reduced level compared to the WT group after Erastin induction.These findings suggest a significant impediment in autophagic vesicle formation, ultimately inhibiting autophagy.
Next, we investigated how autophagy influences the content of LDs and lipid peroxidation levels during ferroptosis.H9c2 cells were treated with 3-MA /RAPA and Erastin for 20 h followed by a 30-min incubation with C11-BODIPY and TPABTBP.As shown in Figure 6c,d, H9c2 cells pretreated with Erastin exhibited a significant increase in lipid peroxidation level and LD content compared to control, indicating the occurrence of ferroptosis.In the presence of both 3-MA and Erastin, the intensity of C11-BODIPY 581/591(representative of lipid peroxidation levels) was significantly reduced compared to the Erastin-induced group (Figure 6d,e).Flow cytometry quantification confirmed a 51.5% decrease in lipid peroxidation levels (Figure 6g).Conversely, the cellular LD content increased by 2.3-fold (Figure 6h).These  6d,f, it is evident that the green fluorescence intensity of C11-BODIPY 581/591 is significantly increased in the Erastin+RAPA group.Flow cytometry quantification results indicate a 1.6-fold increase in lipid peroxidation levels compared to the Erastin group (Figure 6g).On the contrary, in the Erastin+RAPA group, the red fluorescence intensity of TPABTBP (representing LDs) decreased, and quantitative results showed a 29.92% reduction in LD content compared to the Erastin group (Figure 6h).9b,d,38] The above results provide preliminary evidence that inhibiting autophagy reduces LD degradation and alleviates the severity of ferroptosis, whereas promoting autophagy has the opposite effect.
To further confirm the key role of autophagy in regulating LDs and ferroptosis, we monitored LDs and lipid peroxidation levels in WT and ATG5 KO cells after inducing them with Erastin for 20 h.As depicted in Figure S45a (Supporting Information), LDs in ATG5 KO cells significantly increased compared to the WT group after Erastin induction.Quantitative analysis of the average fluorescence intensity in the images revealed a 3.7-fold increase in the red fluorescence intensity of TPABTBP (Figure S45b, Supporting Information).In contrast, the green fluorescence intensity of C11-BODIPY decreased by 80.64% (Figure S45c,d, Supporting Information).These results confirm that LDs are primarily degraded through the autophagy pathway during Erastin-induced ferroptosis.Inhibiting autophagy can impede LD degradation, thereby alleviating lipid peroxidation.However, it is currently unclear which autophagy regulates LD degradation during ischemia/reperfusion-induced cardiomyocyte ferroptosis.
9d,41] Therefore, we employed TPABTBP and BODIPY 493/503 to monitor lipophagy following Erastin pretreatment.As shown in Figure S46 (Supporting Information), in both the 0-h and 9-h groups, the red signal (representing LDs) did not overlap with the green signal (representing lysosomes), indicating that the process of lysosomal engulfment of LDs had not yet occurred.However, after 16 h of Erastin induction, some overlap between Lyso and LDs became visible.After 20 h, lysosomes and LDs almost entirely overlapped.These findings validate the occurrence of LD degradation during ferroptosis via the process of lipophagy.We further performed similar validation experiments using BODIPY 493/503 to label LDs (Figure S47, Supporting Information).Unfortunately, in the 0-h, 9-h, and 20-h groups, there was some co-localization between BODIPY 493/503 and LysoTracker, likely due to nonspecific staining behavior.These findings confirm that LDs are degraded through lipophagy during ferroptosis, and highlight the superior specificity of TPABTBP in imaging LDs compared to BODIPY 493/503.
9b] Therefore, we further investigated whether RAB7A knockdown reduces LD breakdown and suppresses ferroptosis in H9c2 cells (Figure S48, Supporting Information).Polymerase Chain Reaction (PCR) (Figure S49, Supporting Information) and Western blot (Figure S50, Supporting Information) analysis proved that the RAB7A was downregulated in H9c2 cells.As shown in Figure S51 (Supporting Information), the control (Ctrl) siRNA did not significantly affect the number of LDs in RAB7A-knockdown cells.Subsequently, we investigated whether knocking down RAB7A affects lipophagy, thereby regulating LD content and ferroptosis.As shown in Figure S52 (Supporting Information), In the Erastin+RAB7A siRNA group, the red fluorescence signal of LDs and the green fluorescence signal of lysosomes were significantly separated compared to the Erastin-treated group, indicating a marked reduction in their co-localization.These results strongly confirm the occurrence of lipophagy during Erastin-induced ferroptosis in H9c2 cells and demonstrate that downregulating RAB7A effectively inhibits lipophagy.Additionally, confocal FI of TPABTBP showed that compared with normal H9c2 cells treated by Erastin or subjected to H/R, the number of LDs in H9c2 cells with the RAB7A gene knocked down was significantly increased (Figure 6i,j).As shown in Figure S53, upon treatment with Erastin, the MFI of RAB7Aknockdown H9c2 cells was 1.95 times higher than the control group.Moreover, under hypoxia/reoxygenation conditions, the MFI in RAB7A knockdown H9c2 cells exhibited a 1.42-fold increase compared to the control group.This phenomenon indicates that inhibiting the lipophagy pathway can prevent the breakdown of LDs.
We then examined the impact of RAB7A silencing on lipid peroxidation levels under pro-ferroptotic conditions.As shown in Figure S54a (Supporting Information), after 20 h of Erastin induction in H9c2 cells with RAB7A knockdown, lipid peroxidation levels were notably lower compared to cells with normal RAB7A expression.Quantitative analysis showed a 27.2-fold increase in the green fluorescence intensity of C11-BODIPY after Erastin induction, while the fluorescence intensity decreased by 73.38% in the RAB7A knockdown group compared to the normal RAB7A expression group (Figure S54b, Supporting Information).Additionally, RAB7A knockdown reduced the MDA content by 34% and increased the GPX4 content by 5.8-fold compared to the control siRNA group (Figure 6k).Similarly, under H/R conditions, RAB7A knockdown led to a 48.8% reduction in MDA content and a 4.1-fold increase in GPX4 content compared to the control siRNA group (Figure 6l).These findings confirm that inhibiting lipophagy effectively reduces lipid peroxide levels, offering a potential new strategy for mitigating ferroptosis.However, it is worth noting that there are currently and RAB7A-knockdown H9c2 cells were evaluated.j) Assessment of LD content in normal and RAB7A knockdown H9c2 cells under H/R conditions.k, l) The intracellular levels of MDA and GPX4 in three types of H9c2 cells (normal H9c2 cells, RAB7A-knockdown H9c2 cells, and Control siRNA-treated H9c2 cells) were quantified after induction with 5 μM Erastin for 20 h or under H/R conditions.Confocal images were used to measure m) lipid peroxide levels and n) LD content in H9c2 cells treated with culture medium containing Erastin (5 μM) or Erastin +CQ (5 μM) for 20 h.Scale: 50 μm.o, p) The proportion of cells stained with C11-BODIPY 581/591 or TPABTBP in H9c2 cells were analyzed by flow cytometry.Student's t-test to compare statistical differences; *p < 0.05; **p < 0.01; ***p < 0.001.
no commercially available inhibitors specifically targeting lipophagy.
To validate our findings, we treated Erastin-induced H9c2 cells with chloroquine (CQ), a well-established autophagy inhibitor, [42] for 24 h, and then assessed the autophagy flux.As shown in Figure S55 (Supporting Information), in the Erastin/CQ group, there was a significant increase in the number of yellow fluorescent puncta (autophagosomes), while the number of red fluorescent puncta (autolysosomes) markedly decreased.This indicated that CQ disrupted the fusion between autophagosomes and lysosomes, thereby impeding autophagy at the stage of autophagosome formation. [39]Subsequently, the cells were incubated with C11-BODIPY or TPABTBP to assess the extent of lipid peroxidation and the content of LDs (Figure S56, Supporting Information).Upon addition of CQ to Erastin-treated H9c2 cells, the intensity of green fluorescence emitted by C11-BODIPY significantly decreased (m).Flow cytometry analysis further demonstrated that the lipid peroxidation level in cells following CQ treatment decreased from 29.16% to 16.1% compared to the Erastin-treated group (o).However, under the same conditions, the LD count exhibited a contrasting trend.In the presence of CQ, Erastin-treated H9c2 cells showed a notable increase in cytoplasmic LDs (Figure 6n), as confirmed by flow cytometry, with LD count being 1.9-fold higher compared to the Erastin group (Figure 6p).Taken together, these findings suggest that CQ inhibits LD degradation and protects cardiomyocytes from lipid peroxidation-induced damage.

Evaluation of the Biosafety of the Probe TPABTBP
We conducted comprehensive assessments to evaluate the safety profile of TPABTBP both in vitro and in vivo.Initially, we examined the cytotoxic effects of TPABTBP on H9c2 cells across a range of concentrations (5, 10, 30, and 120 μM) using CCK8 assays.Remarkably, TPABTBP exhibited no discernible toxicity even at the highest concentration tested (120 μM), as evidenced by the cell viability data (Figure S57, Supporting Information).Subsequently, we investigated the impact of TPABTBP on red blood cells (RBCs) via hemolysis assays.RBCs isolated from C57BL/6J mice were exposed to various concentrations of TPABTBP (0, 5, 10, 30, and 120 μM), along with Triton X-100 [43] as a positive control.Notably, TPABTBP did not induce any hemolysis within the tested concentration range (0-120 μm), as demonstrated by the absence of significant hemoglobin release (Figure S58, Supporting Information).
Moving on to in vivo evaluations, mice were administered TPABTBP (2 mg kg −1 ) via intravenous injection every 48 h for three consecutive doses.Control group mice received PBS solution injections under identical conditions.The safety profile was assessed by analyzing serum levels of liver and kidney function markers, including alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine (CREA), and blood urea nitrogen (BUN).Importantly, there were no notable differences in hematological parameters or liver and kidney function between the TPABTBP-injected group and the control group (Table S5 and Figure S59, Supporting Information).Furthermore, histological analysis of major tissues (kidney, spleen, heart, liver, and lungs) collected seven days after intravenous TPABTBP administration (2 mg kg −1 ) revealed no signs of necrosis, inflammation, hemorrhage, or other abnormalities.Hematoxylin and Eosin (H&E) staining confirmed the absence of tissue damage or pathological changes (Figure S60, Supporting Information).
These results demonstrated that TPABTBP exhibited negligible in vivo toxicity and excellent safety.

Imaging the Dynamic Changes of LDs in I/R Injured Mice
In vitro experiments have confirmed that cardiomyocyte I/R injury can induce ferroptosis, a process accompanied by dynamic alterations in LD numbers.To delve deeper into the regulation of LDs on myocardial cell ferroptosis during I/R injury, we established a mouse model of MIRI. [6,44]The results of the triphenyltetrazolium chloride (TTC) assay revealed distinct myocardial ischemic areas following 30 min of ischemia and 24 h of reperfusion in mice subjected to MIRI compared to the shamoperated group (Figure 7a).Histological examinations, including hematoxylin and eosin (H&E) and Masson's staining, as well as myocardial enzyme analysis, further confirmed the successful induction of MIRI.The myocardial tissue from the I/R group exhibited irregular shape, disordered arrangement, abnormal intercellular spacing, and inflammatory cell infiltration, along with myocardial fibrosis indicated by blue-colored collagen-enriched areas in Masson's trichrome staining (Figure 7b,c).Moreover, biochemical markers such as aspartate transaminase (AST), creatine kinase (CK), creatine kinase-MB isoenzyme (CK-MB), and lactate dehydrogenase isoenzyme 1 (LDH1) were significantly elevated in the serum of mice subjected to MIRI compared to the sham group, confirming successful model establishment (Figure 7d-g).
Subsequently, we evaluated lipid peroxidation levels in myocardial tissue to investigate ferroptosis induction during MIRI.As shown in Figure 7h, MIRI led to a significant increase in lipid peroxidation levels compared to the sham group.Immunofluorescence assays revealed a notable decrease in glutathione peroxidase 4 (GPX4) protein expression after 24 h of reperfusion, further indicating ferroptosis induction (Figure 7i).Prussian blue staining with diaminobenzidine (DAB) revealed a marked accumulation of nonheme iron in myocardial tissues after 24 h of reperfusion, with a 4.5-fold increase compared to the sham group (Figure 7j,k).To further validate ferroptosis induction, we measured the levels of ferroptosis-associated markers (GPX4, 4-HNE, and MDA) in blood serum. [45]As shown in Figure 7l, after 24 h of myocardial reperfusion, the level of GPX4 in the serum was significantly lower than that in the sham group (19.17 vs 25.93 mU mg −1 ).Compared with those in the sham surgery group, the levels of 4-HNE and MDA in the I/R group increased 1.8-and 1.3-fold, respectively (Figure 7m,n).TEM imaging of myocardial tissue showed that the mitochondrial membrane densities were condensed in I/R injury mice, a typical feature of ferroptosis [46] (Figure 7o).These results strongly confirm that myocardial I/R in mice damages the myocardium by inducing ferroptosis, which is consistent with the results of previous studies. [6,47]ext, we used the probe TPABTBP to image cardiomyocyte LDs during MIRI.Initially, we conducted in vivo biodistribution studies of TPABTBP to determine suitable imaging time points.Subsequently, fluorescence imaging and quantitative analysis on the dissected hearts of mice injected with TPABTBP (2 mg kg −1 ).The results, as depicted in Figure S61a,b (Supporting Information), revealed that cardiac fluorescence intensity at 6 h postinjection was 2.5 and 1.7 times higher than at 1 and 3 h, respectively, with no significant difference observed between 6 and 12 or 24 h.These findings suggested that a 6 h interval was optimal for acquiring high-quality cardiac imaging results.Furthermore, quantification of fluorescence signals in major organs (heart, lungs, liver, spleen, kidneys) indicated a robust signal of TPABTBP in the liver and kidneys (Figure S61c, Supporting Information).This observation aligns with the metabolic pathway of small molecules, primarily metabolized in the liver and excreted through the kidneys following intravenous injection. [48]e then utilized TPABTBP to track the LD content in myocardial tissue at different perfusion time points.In the experiment (Figure 8a), mice underwent myocardial I/R and received intravenous injections of the TPABTBP probe (2 mg kg −1 ) after 0, 3, 6, and 18 h of reperfusion.After 6 h, their hearts were imaged ex vivo.To mitigate background fluorescence from the heart, mice in the sham operation group were also intravenously injected with the same amount of TPABTBP as controls.The imaging results revealed weak fluorescence in the hearts of sham-operated mice.However, compared to the sham group, fluorescence intensity increased by 1.9-fold after 6 h of reperfusion, further rising to 3.5fold after 9 h.Yet, this intensity decreased by 18.26% after 12 h compared to that at 9 h, followed by a 37.4% reduction after 24 h (Figure 8b,c).These results firmly confirm a significant increase in the number of LDs during the early stage (9 h) of myocardial I/R, with substantial decomposition occurring in the later stage of reperfusion.This observation aligns with previous in vitro cell experiments.
To verify that the specificity of the fluorescence signal in the heart was due to the binding of the probe TPABTBP to LDs, we froze and sliced the heart after imaging and observed it using confocal microscopy.As shown in Figure 8d, the LDs in the 24 h group were markedly reduced compared to those in the 9 h group.The GPX4 level in the 9 h group was higher than that in the 24 h group, suggesting that an increase in the number of LDs may exert an antagonistic effect on ferroptosis (Figure 8e).Oil red O staining of myocardial tissue confirmed that the number of LDs in the myocardial tissue increased significantly after 9 h of ischemia treatment but decreased after 24 h (Figure 8f).These findings suggest a strong correlation between MIRI and ferroptosis, with LD degradation promoting ferroptosis.During the early reperfusion stage, LD numbers in myocardial tissue notably rise, serving as reservoirs for FFAs, thereby inhibiting lipid peroxidation.However, during the later reperfusion stage, ferroptosis becomes inevitable, and LDs undergo extensive decomposition, releasing FFAs, which promote lipid peroxidation and exacerbate myocardial injury.Therefore, inhibiting LD decomposition during the early reperfusion stage may attenuate MIRI.

Chloroquine (CQ) Inhibits LD Degradation to Mitigate MIRI
As previously demonstrated, CQ effectively inhibited LD decomposition in H9c2 cells, thereby mitigating ferroptosis.Subsequently, we sought to determine whether CQ could attenuate MIRI in mice.For early intervention, mice received intraperitoneal injections of CQ (30 mg kg −1 ) 1 h after reperfusion [8b,49] (Figure 9a).Serum and myocardial tissue were collected 24 h and 4 weeks post-reperfusion to evaluate the therapeutic efficacy.In comparison to the I/R group, the CQ-treated group exhibited a significant reduction in myocardial infarct size, as evidenced by TTC staining (Figure S62, Supporting Information).Additionally, myocardial fibrosis was notably diminished after CQ treatment (Figure 9b).Interestingly, nonheme iron levels were reduced by 70% in myocardial tissue after CQ treatment compared with that in the untreated group (Figure 9c,d).Similarly, after CQ treatment, serum 4-HNE levels in mice were reduced by 43% compared with that in untreated groups (Figure 9e).These findings suggest that CQ effectively inhibits ferroptosis induced by myocardial I/R.We then investigated whether this protective effect was conferred to LD degradation inhibition.The probe TPABTBP and Oil red O were used to stain LDs in cardiac tissue after I/R.As shown in Figure 9f, the number of LDs in the myocardial tissue of mice after 24 h of I/R was significantly decreased, while the number of LDs was significantly increased after CQ treatment.Moreover, the extensive colocalization of the red fluorescence signal from TPABTBP with that of BODIPY 493/503 confirmed the suitability of TPABTBP for specific LD imaging in myocardial tissue (Figure S63, Supporting Information).Oil red O staining of myocardial tissue indicated the increase in LD number in myocardial tissue after CQ treatment (Figure 9h).Immunofluorescence staining of GPX4 showed significantly higher GPX4 expression in the myocardium after CQ treatment than that in the untreated group (Figure 9g).The increased expression of GPX4 indicated that CQ treatment inhibited LD decomposition, thereby improving the ability of cardiomyocytes to resist lipid peroxidation and attenuate ferroptosis-induced myocardial injury.To observe the ultrastructural changes in myocardial tissue after treatment, we performed TEM to examine the hearts of CQ-treated mice.Our analysis revealed that I/R-treated mice exhibited severe mitochondrial distortion and vacuolation, with numerous autophagic vesicles appearing in the cytoplasm.Then, CQ treatment mitigated the effects of I/R induction (Figure 9i).Moreover, the levels of myocardial enzymes (AST, CK, CK-MB, and LDH) in serum, which increased due to I/R, were significantly reduced in mice treated with CQ (Figure 9j-m).Taken together, these results suggest that CQ exerts a therapeutic effect on MIRI by inhibiting ferroptosis.We hypothesize that CQ may reduce LD degradation and mitigate ferroptosis by inhibiting lipophagy, [41a] thereby alleviating MIRI injury.However, the mechanism underlying this effect remains to be further elucidated.
Subsequently, we examined the biosafety of CQ in vivo.The serum and major organs of the mice were collected for evaluation 24 h after intraperitoneal injection of 30 mg kg −1 ) CQ.As shown in Figure S64 (Supporting Information), levels of the myocardial enzymes (AST, CK, CK-MB, and LDH1) in the CQ-treated group did not significantly differ from those of the control, confirming the absence of significant cardiotoxicity induced by TPABTBP.Furthermore, serum levels of ALT, BUN, and CREA were within normal ranges, suggesting unaffected liver and kidney metabolism in the mice (Figure S65, Supporting Information).Additionally, H&E staining of major organs revealed structurally intact cardiomyocytes with well-defined and regular  nuclei.No significant pathological damage was observed in the livers, spleens, or kidneys (Figure S60, Supporting Information).Taken together, these results demonstrate that CQ shows good biosafety and is a prospective medication for treating MIRI.

Conclusion
In summary, we developed an LD-activated AIE probe, TPABTBP, for monitoring the dynamic changes in LD number during myocardial I/R-induced ferroptosis in mice.The probe TPABTBP showed excellent LD-targeted imaging ability and good biocompatibility.MD simulations and super-resolution FI confirmed that the probe TPABTBP was specifically localized to the phospholipid monolayer membrane of LDs.Compared to the commercially available LD fluorescence dye BODIPY 493/503, TPABTBP demonstrated superior LD specificity, enhanced capability for monitoring lipophagy, and remarkable photostability.Using TPABTBP as an imaging probe revealed that LD accumulation in cardiomyocytes was increased during the early stage of ferroptosis but decreased via lipophagy in the late stage.Inhibition of autophagy or knockdown of the lipophagyrelated gene RAB7A led to a reduction in the decomposition of LDs and conferred protection against ferroptosis damage onto cardiomyocytes.Imaging of myocardial I/R injured mice with TPABTBP showed that the LD content in myocardial tissue increased significantly after 9 h of reperfusion and decreased after 24 h.More importantly, we confirmed that CQ inhibited LD decomposition to alleviate MIR-induced ferroptosis in vivo.However, whether the inhibitory effect of CQ on ferroptosis is mediated through mechanisms associated with lipophagy requires further investigation.
position of the headgroup plane in the phospholipid bilayer.The headgroup plane was determined by calculating the average position of phosphorus atoms along the z-axis.
To analyze the free energy landscape of TPABTBP migration through the two interfaces, a multi-walker well-tempered meta-dynamics simulation was conducted with 4 replica walkers.For TPABTBP, four initial representative structures were selected from unrestrained MD simulation trajectories, each with varying migration depths ranging from −2 to −0.5 (−2 ≤ z ≤ −0.5).In each of these four simulations, TPABTBP was initially placed within the phospholipid layer.We employed two collective variables (CV) to characterize the migration depth (z) of TPABTBP and the angle.The angle was defined as the angle between the R end to end vector and the membrane normal.The parameters of meta-dynamics were set as follows: Gaussians were deposited at intervals of 500-time steps with an initial height of 1.2 kJ mol −1 .A bias factor of 5 was judiciously chosen.The Gaussian width (sigma) was determined based on the fluctuation of the collective variables observed during the unbiased run and set at 0.2.The system was maintained at a temperature of 300 K.And other parameters were defaults.The total time of meta-dynamics simulation was 5 ns for each initial representative structure.
Isolation and Visualization of Lipid Droplets (LDs) in HepG2 Cells: HepG2 cells (3 × 10 7 ) were initially treated with trypsin and subsequently resuspended in a complete growth medium.The cell suspension was then subjected to centrifugation at 1000 g for 5 min.After centrifugation, the culture medium was carefully removed.These cells were washed with 10 mL PBS, and centrifuged again at 1000 g for 5 min.Subsequently, the cell pellet was resuspended in 1 mL of PBS.The resuspended cells were transferred into a 2 mL Eppendorf (EP) tube, and centrifuged again at 1000 g for 5 min.The supernatant was then removed.Next, the pellet was thoroughly resuspended in 200 μL of Reagent A and incubated on ice for 10 min.Carefully, 800 μL of 1×Reagent B was added to the mixture and thoroughly mixed.After incubating on ice for 10 min, the cells were homogenized by passing them through a 27-gauge needle attached to a 3 mL syringe five times.Subsequently, briefly centrifuge the homogenate at 100 g for 5 s.Finally, 600 μL of 1× Reagent B was layered on top of the homogenate.The EP tube containing the layered components was subjected to centrifugation in a microcentrifuge for 3 h at a force ranging from 18 000 to 20 000 g at a temperature of 4 °C.After centrifugation, 270 μL of floating LDs from the top of the EP tube were collected.These LDs were subsequently transferred for storage at −80 °C.
Subsequently, disperse 10 μL LDs in 0.5 mL cyclohexane and subject the mixture to ultrasonic dispersion for a duration of 10 min.Following this step, incubate the resulting LD solution with 1 mM TPABTBP at room temperature for a period of 3 min.Immediately, transfer 10 μL of the resulting mixture onto a glass slide and proceed to observe it under a multispectral fluorescence microscope.
Moreover, HepG2 cells were seeded in confocal dishes, followed by staining with 30 μM TPABTBP for 0.5 h.Next, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and then labeled with a DAPI staining solution for nuclear identification.Finally, LDs within the HepG2 cell cytoplasm were observed using a 100× oil immersion lens on a multi-color fluorescence laser confocal microscope (FV3000, Olympus, Japan).
Co-localization Analysis of GFP-tagged PLIN2 and TPABTBP: Initially, 5 × 10 4 HepG2 cells were seeded into confocal dishes and cultured overnight at 37 °C with 5% CO 2 .Subsequently, GFP-tagged PLIN2 was diluted in Opti-MEM medium, and HepG2 cells overexpressing GFP-tagged PLIN2 were constructed via transfection according to the manufacturer's instructions using Lipo3000.Following this, HepG2 cells were co-cultured with 30 μM TPABTBP for 0.5 h.The HepG2 cells were then washed with PBS, fixed with 4% paraformaldehyde, and finally washed three times with PBS.The LDs within the HepG2 cells were observed using a laser confocal microscope (FV3000, Olympus, Japan).
In Situ Time-Lapse Confocal Imaging of LDs: HepG2 cells (1 × 10 4 ) were seeded into a confocal dish and cultured at 37 °C with 5% CO 2 for 12 h.Subsequently, the cells were incubated with 30 μM TPABTBP staining solution without serum for 0.5 h.Following incubation, the cells were rinsed with PBS containing 1% FBS.The confocal dish was then placed in a live-cell workstation.The observation of LDs was conducted using a comprehensive spectral ultra-high resolution laser confocal scanning system, which performed a layered scan across five Z-positions on HepG2 cells (Stellaris STED, Leica, Germany).
Structured Illumination Microscopy (SIM) Imaging of LDs: H9c2 and HepG2 cells (1 × 10 4 per slide) were seeded into glass cover slides and cultured for 12 h at 37 °C with 5% CO 2 .LDs in H9c2 cells were labeled by incubating them with TPABTBP (30 μM) and BODIPY 493/503 (5 μM) in serum-free medium at 37°C in the dark, and HepG2 cells were also labeled by incubating them with TPABTBP (30 μM).After 0.5 h of incubation, the treated cells were washed 3 times with PBS and preserved in ProLong Gold antifade reagent.Finally, the LDs were observed under a Lattice SIM 2 ZEISS Elyra 7 microscope. [58]esponsiveness of Different Probes to Cellular LDs: HeLa, RAW264.7, and H9c2 cells were seeded at 1 × 10 4 cells mL −1 into confocal dishes.After 12 h in culture, the medium was removed, and the cells were coincubated with PMBTDP (10 μM), BTDPP (10 μM), TPABTBP (10, 30 μM) and BODIPY 493/503 (5 μM) for 0.5 h.The cells were then washed three times with PBS in the confocal culture dish and fixed using a 4% paraformaldehyde solution.DAPI staining was performed for 10 min.Then, images were captured using a Zeiss LSM 780 single-photon laser confocal scanning microscope, and fluorescence colocalization was analyzed using ImageJ software.
Immunofluorescence Staining: H9c2 cells were initially seeded onto glass coverslips and incubated overnight at 37 °C under 5% CO 2 conditions.Subsequently, the cells were treated with 30 μM TPABTBP for 30 min.Afterward, the cells were washed three times with PBS and fixed in 4% paraformaldehyde at room temperature for 10 min.Following fixation, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 5 min.H9c2 cells were then washed three times with PBS for 5 min each.Subsequently, the cells were treated with QuickBlock immunostaining blocking solution for 10 min.For overnight incubation at 4 °C, cells were treated with Rabbit monoclonal antibody to PLIN2, following the manufacturer's instructions.Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H+L) was then added to the cells for visualization.DAPI was used for nuclear counterstaining.Finally, images were obtained using a confocal microscope.
HepG2 cells (5 × 10 4 cells mL −1 ) were seeded into 35 mm glass-bottom culture dishes and incubated for 12 h at 37 °C with 5% CO 2 .After the incubation period, cells were treated with 1 mL LDs, Mito, ER, GA, or Lyso tracker work solution for 30 min.Following probe incubation, cells were washed three times with HBSS buffer.Subsequently, HepG2 cells were treated with 30 μM TPABTBP for 30 min.Finally, cells were subjected to confocal microscopy analysis.Image analysis was performed using Im-ageJ.
Furthermore, H9c2 cells (5 × 10 4 cells mL −1 ) were seeded into 35 mm glass-bottom culture dishes and incubated overnight at 37 °C with 5% CO 2 .Subsequently, the cells were treated with 1 mL of 75 nM LysoTracker Red DND-99 for 30 min.Afterward, the cells were washed three times with HBSS buffer, followed by treatment with 30 μM TPABTBP or 5 μM BOD-IPY493/503 for 30 min.Finally, the cells were stained with DAPI for 10 min and subjected to confocal microscopy analysis.
Detection of Cellular LDs after Ferroptosis Induction: Hela, H9c2, and HepG2 cells (1 × 10 4 cells per well) were treated with 5 μM Erastin in a growth medium for 1, 3, 6, 9, 12, 16, 20, and 24 h.These cells were incubated with TPABTBP (30 μM) in confocal dishes or 6-well plates at 37 °C for 0.5 h.Subsequently, the cells in the 6-well plate were washed three times with PBS before being detached from the dish by scraping.Next, these cells were washed with PBS, fixed with paraformaldehyde, and stained with DAPI.Confocal fluorescence scanning microscopy (CLSM) was then performed to obtain fluorescence images.The intensity of LD staining was quantified by measuring the mean fluorescence intensity (MFI) within cells.MFI was quantified in individual cells using ImageJ (Fiji 2.15.0), and n = 3 for each measurement.
Besides, H9c2 cells were further incubated with TPABTBP (30 μM) in 6-well plates at 37 °C for 0.5 h.Subsequently, the cells were washed three times with PBS before being detached from the well by scraping.H9c2 cells were resuspended in 300 μL of PBS and used for flow cytometry measurements (Alex488 channel, SONY ID7000 full spectrum analysis flow cytometer).
In addition, H9c2 cells were stained with Oil Red O and the intracellular distribution of the LDs after Erastin treatment was observed at different time points (3, 6, 9, 12, and 24 h).First, the post-induced H9c2 cells were fixed with 4% paraformaldehyde.Subsequently, they were covered with 60% isopropanol solution and allowed to incubate for 15 s.Following this, the isopropanol solution was removed, Oil Red O was introduced, and the cells were immersed in the dye for a period of 0.5 h.Next, the cell nuclei were stained with hematoxylin for 3 min.The cells were counterstained with aniline blue and then washed with water.Finally, the LDs were photographed with a Nikon Ni-E optical microscope after a drop of glycerol-gelatin was applied to seal the sample.
Lipid Peroxidation Assay: H9c2 cells were seeded in a 6-well plate or confocal dish (1 × 10 4 cells mL −1 ).C11-BODIPY 581/591 was dissolved in dimethyl sulfoxide (DMSO) to produce a 5 μM working solution.In the reduced state, the excitation and emission maxima of C11 BODIPY 581/591 was 581/591 nm; after oxidation, the probe shifted the excitation and emission to 488/510 nm.The cells were then collected and incubated with this C11-BOPIPY 581/591 working solution for a total of 0.5 h after treatment with Erastin for 3, 6, 9, 12, or 24 h.For cell counting by flow cytometry (in the BODIPY channel, E ex = 488 nm), the H9c2 cells in the 6-well plate were removed and suspended in 0.3 mL of PBS.After fixing and staining with DAPI, the cells were analyzed by CLSM.
Hypoxia/Reoxygenation (H/R) Cell Culture: To simulate MIRI in vitro, a hypoxic culture system was constructed according to the AnaeroPack (Mitsubishi, Japan) instructions.In brief, culture bags, disposable anaerobic oxygen capsules, oxygen indicators, and airtight containers were used.H9c2 cells were cultured overnight at 37 °C with 5% CO 2 until the cells adhered to the walls of culture dishes with the oxygen concentration at 0.1% and the CO 2 level at 5% for 1 h in the cell culture system.After 12 h of anoxic incubation, the dishes with H9c2 cells were removed from the closed vessel and placed in a reoxygenation system for culturing at 37 °C for 2 h.Finally, the experiment was terminated, and the cells were labeled with C11-BODIPY 581/591 (5 μM) and TPABTBP (30 μM) for 0.5 h, fixed and washed.The cells were then observed by CLSM.
Calcein-AM /PI Staining: H9c2 cells were seeded in 96-well plates or confocal culture dishes (1 × 10 4 cells mL −1 ).Subsequently, a detection buffer was utilized to dilute the Calcein-AM and propidium iodide (PI) staining solutions by a factor of 1000, creating staining working solutions.Following hypoxia-reoxygenation cultivation or induction with Erastin for 3, 6, 9, 12, or 24 h, cells were collected and washed once with PBS.The combined Calcein-AM and PI working solutions were then applied to the cells, followed by incubation in the dark for 0.5 h.After staining, detection was performed using laser confocal microscopy or a fluorescence microplate reader.(Calcein-AM excitation/emission = 494/517 nm; PI excitation/emission = 535/617 nm).
Transmission Electron Microscopy (TEM): The cells and tissues were exposed to a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 2 mM CaCl 2 in a 0.15 m sodium calcium carbonate buffer solution at room temperature for a duration of 2 h.Then, the specimens were fixed in a 1% osmic acid solution for another 2 h.The samples were washed three times with 0.1 M PBS, sliced, and dehydrated with acetone.Epoxy 812 (Ted Pella) was used to embed the specimens.After polymerization of the specimens in an oven at 60 °C for 48 h, ultrathin sections (70 nm) were prepared under an ultramicroscope (Leica EM UC7) with a diamond sectioning knife (Ultra 45°) and placed on copper mesh (300 mesh).Uranium-lead double-stained sections were imaged with a Hitachi HT7700 TEM system operating at 80 kV.
Inhibition of Cellular Autophagy: H9c2 cells were cultured with 5 μM Erastin and 100 nM rapamycin (RAPA) or 10 mM 3-methylladenine (3-MA) for 20 h.The H9c2 cells were then labeled with 30 μM TPABTBP or 5 μM C11-BODIPY 581/591.After 0.5 h of incubation, the cells were fixed with 4% paraformaldehyde and labeled with DAPI.Subsequently, the H9c2 cells were observed by fluorescence microscopy, and the percentage of positive cells was determined based on the flow cytometry data.
Generation and Validation of Knockout ATG5 Cell Line: For the generation of ATG5 knockout (KO) cells, single guide RNAs (sgRNAs) were designed using the online CRISPR design tool (Red Cotton, China).Specifically, sgRNA-1 (g5) had the sequence GTGATAGGTGTGCGGAAGTTGG, and sgRNA-2 (g6) had the sequence AGAACTCTACTCCGTGGTTTAGG (Table S3, Supporting Information).These sgRNAs were designed to target the exon region of the ATG5 gene for CRISPR/Cas9 genome editing.A prioritized list of sgRNAs was generated, taking into account both specificity and efficiency scores.Oligos corresponding to the two targeting sites were annealed and ligated into the YKO-RP006 vector (Ubigene Biosciences Co., Ltd., China).The resulting YKO-RP006-hRABL6[gRNA] plasmids containing the target sgRNA sequences were transfected into the target cells using Lipofectamine 3000 (Thermo Fisher Scientific).Subsequently, 24-48 h after transfection, puromycin was introduced to facilitate the selection of successfully edited cells.Following antibiotic selection, a specific number of cells were subjected to limited dilution and subsequently inoculated into a 96-well plate.Single clones were selected after 2-4 weeks of culture.
In addition, these ATG5 KO clones were subsequently validated using PCR and Sanger sequencing.The genomic DNA from H9c2 cells was extracted using the genomic DNA extraction kit.Simultaneously, primers were designed to target the knockout region for PCR amplification.The nucleic acids were subjected to denaturation, annealing, and extension steps, followed by further analysis of the target gene's knockout status through gel electrophoresis.Furthermore, PCR products were subjected to TA cloning, followed by Sanger sequencing to definitively determine the mutation status of each allele.The sgRNAs and primer sequences employed for PCR design can be found in Table S4 (Supporting Information).
Detection of Autophagy Flux: H9c2 cells and ATG5 KO cells were seeded in a 6-well plate and allowed to achieve 50%−70% confluence before transfection.According to the manufacturer's instructions, cells were infected with mCherry-eGFP-LC3 adenovirus in the culture medium for 24 h.Subsequently, the culture medium was replaced with a fresh complete medium, and the cells were further incubated at 37 °C for an additional 24 h.Following this incubation period, the H9c2 cells were treated with a combination of RAPA (100 nM), 3-MA (10 mM), or CQ (5 μM), in addition to 5 μM Erastin, and incubated further.Finally, images were acquired using laser confocal microscopy, and ImageJ was used to count yellow puncta (autophagosome) and red puncta (autolysosome).
qPCR: Total mRNA was extracted from H9c2 cells with RAB7A knockdown, as well as from cultured cells, using TRIzol reagent.Subsequently, cDNA synthesis was carried out employing reverse transcription reagents from Vazyme.qPCR was conducted using PerfectStart Green qPCR Super-Mix (+Dye II), following the manufacturer's recommended protocols.The mRNA expression levels of the relevant genes were normalized to the expression of the housekeeping gene -actin.
WB: Total protein was extracted and lysed from the RAB7A downregulation H9c2 cells using RIPA lysis buffer.Protein concentrations were measured using the BCA protein assay kit.Equal amounts of the specified proteins were loaded onto a 10% SDS-PAGE gel and subsequently

Measurement of the LD Content in Myocardial Tissue: Oil Red O Staining:
The saturated Oil Red O solution prepared with isopropanol was mixed with distilled water in a 6:4 ratio.This solution was placed in a 60-70°C water bath for 0.5 h, cooled naturally, and filtered through qualitative filter paper to obtain an Oil red O working solution.The frozen myocardium sections were removed from the −20 °C freezer, allowed to stand at room temperature for 5-10 min to reach room temperature, and gently immersed in working solution for 8-10 min.The slices were removed from the solution, allowed to stand for 3 s, and then submerged in two cylinders containing 60% isopropanol for 3-5 s.The slices were then stained with hematoxylin, washed, and then sealed with glycerol-gelatin.Photomicrographs were captured using an Eclipse microscope (Nikon Ni-E).
LD Staining: Frozen myocardial sections were treated with BTDPP, PMBTDP, and TPABTBP for 0.5 h respectively, and the cells were labeled with DAPI after fixation.Finally, laser confocal microscopy imaging was performed to determine the LD content through ImageJ.
In Vitro Fluorescence Imaging (FI) of the Heart: For evaluating the biodistribution of TPABTBP, mice (n = 3 per independent experiment) were injected with TPABTBP (2 mg kg −1 ) via the tail vein.Following this, the mice were sacrificed and dissected at 1, 3, 6, 12, and 24 h postinjection.Main organs (heart, liver, spleen, lungs, and kidneys) were removed for in vitro FI on an IVIS spectrum system (PerkinElmer, USA).
To assess the LD content, model mice of MIRI (n = 5 per independent experiment) were injected with TPABTBP (2 mg kg -1 ) through the tail vein after reperfusion for 0, 3, 6, and 18 h.They were sacrificed 6 h later, and the hearts were removed for in vitro FI on an IVIS spectrum system.The sham operation group received the same dose and was imaged 6 h later.We then sliced each heart immediately into 4-5 short-axis sections and imaged them again.
In all FI experiments, fluorescence reflectance images were obtained employing 480 nm excitation and 670 nm emission filter settings, with a 6 s exposure time.White light images were acquired for each FI dataset, and the fluorescence signal was quantified using Living Image (version 4.4).
Biosafety in Vitro: First, 6×10 4 H9c2 cells per well were uniformly inoculated in 96-well plates, and then, different concentrations of TBABTBP (0, 5, 10, 30, and 120 μM) diluted with DMEM containing 10% FBS was added.These cells were cocultured with cardiomyocytes in a volume of 0.1 mL per well for 12 h.Then, CCK8 reagent (HyClone, China) was added and incubated with the cells for 1.5 h.The final OD580 values were read using an enzyme marker.The percentage of viable cells was calculated, and the difference in cell viability between the treatment and control groups was determined.In addition, 200 μL of erythrocytes were extracted from healthy mice and placed these cells onto plates.Then, the erythrocytes were cultured with PBS (negative control), Triton-X100 (positive control), and various concentrations of TPABTBP at 37 °C for 6 h.The cells were then centrifuged, and the supernatants from each sample were analyzed by determining the OD at 414 nm using a multifunctional microplate detection platform (TECAN SPARK, Switzerland).The hemolysis rate (%) was calculated as follows: hemolysis rate (%) = (OD sample-OD negative)/ (OD positive-OD negative) × 100%.We also collected 1 mL of erythrocytes from healthy mice; diluted the cells in saline at a ratio of 1:3; dispersed them in Eppendorf (EP) tubes; mixed them with PBS, Triton X-100, or probes; and incubated them for 2 h before obtaining images.
Biosafety in Vivo: C57BL/6J mice were randomly assigned to a PBS, TPABTBP (2 mg kg −1 ), and CQ (30 mg kg −1 ) group.The mice in the PBS and TPABTBP groups (n = 5) were injected in the tail vein every 48 h intervals three times.On day 7, blood was collected from the mice, which were then sacrificed and dissected.Samples were collected for routine H&E evaluation of the kidney, liver, heart, spleen, and lungs.The mice in the CQ group were given a single intraperitoneal injection, sacrificed, and dissected the next day.The processes performed with the CQ group were the same as those performed with the other two groups.A portion of the collected whole blood was used for routine blood tests, while the other portion was centrifuged, and the serum was collected to evaluate the AST, urea nitrogen (BUN), creatinine (CREA), and alanine aminotransferase (ALT) levels using the respective assay kits.
Statistical Analysis: Data analysis and graph preparation were performed using GraphPad Prism software.All the summarized data were presented as the mean ± standard deviation (SD).Blinding or randomization was used.Student's t-tests were performed to compare two groups and one-way ANOVAs were performed for comparisons among three or more groups.Differences were considered to be significant when the pvalue was < 0.05.

Scheme 1 .
Scheme 1. Schematic diagram of membrane-targeting aggregation-induced emission probe for monitoring lipid droplet (LD) dynamics in ischemia/reperfusion-induced cardiomyocyte ferroptosis.The LD-targeting molecular probe TPABTBP was trapped in the phospholipid monolayer of LD.TPABTBP was employed to monitor the dynamics of LD during MIRI, indicating the involvement of LD degradation through lipophagy.Meanwhile, inhibiting lipophagy has been shown to protect cardiomyocytes from ferroptosis.

Figure 1 .
Figure 1.The design strategy of the proposed LD probes and their photophysical properties.a) Polarity responsiveness and intramolecular motion restriction were leveraged to design probes with aggregation-induced emission for use in LD imaging.The fluorescence emission spectra of b) TPABTBP, c) PMBTDP, and d) BTDPP in different solvents.Amplitude plots of highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) energy levels of e) TPABTBP, f) PMBTDP, and g) BTDPP as calculated by using the PBE0/Def2-svp basis set.Photoluminescence (PL) spectra of h) TPABTBP, j) PMBTDP, and l) BTDPP in DMSO/water mixtures with different water fractions (f w ).Plots showing maximum PL intensity of i) TPABTBP, k) PMBTDP, and m) BTDPP at various DMSO/water mixtures.Inset: photographs of TPABTBP, PMBTDP, or BTDPP fluorescence in DMSO solution (left) and DMSO/water mixtures (right) with a 95% water content.A hand-held UV lamp was used to obtain these photographs.Concentration: 10 μM;  ex = 480 nm.

Figure 3 .
Figure 3. Molecular dynamics (MD) simulation of probe TPABTBP migration across the cytosol to the LD phospholipid monolayer.a) Representative snapshots showing TPABTBP migration to the phospholipid monolayer during a 500 ns simulation.b) End-to-end TPABTBP migration distance (R end-to-end ).c) The position of the migrating TPABTBP in relation to the phospholipid monolayer was determined, with the phospholipid headgroup fixed at z = 0 and the monolayer range highlighted in yellow-green.d) The free energy landscape of TPABTBP migration into LDs was determined using multi-walker well-tempered meta-dynamics simulations, and the representative structures are illustrated in the inset figures, with each image linked to the migration depth and angle (between the R end to end vector of TPABTBP and the membrane normal).

Figure 4 .
Figure 4. Structured illumination microscopy (SIM) imaging of LDs in live cells.a) SIM images of H9c2 cells stained with 30 μM TPABTBP for 0.5 h.b,c) Corresponding enlarged SIM images at different scales (2 μm, 200 nm).d-f) Representative images and fluorescence intensity profiles of the LDs by SIM 2 (white arrow: diffraction limit).g) SIM images of LDs in HepG2 cells stained with 30 μM TPABTBP for 0.5 h.Scale bar: 2 μm.Corresponding enlarged SIM images h, i) at different scales (1 μm, 500 nm).j-l) Reconstruction of z-axis images from SIM viewed from different angles.

Figure 6 .
Figure 6.Detection of LDs and ferroptosis in H9c2 cells after inhibition of lipophagy.a,b) Transmission electron microscopy (TEM) images of H9c2 cells treated with 5 μM Erastin for 24 h (black arrows: autophagic vacuole; *: LD).Lipid peroxidation level and LD content in H9c2 cells were divided into four groups: the c) untreated group, the d) Erastin-induced group, the e) Erastin + 3-MA treated group, and the f) Erastin + RAPA treated group.Green and red channels correspond to lipid peroxides and LDs.Reagents: 5 μM Erastin; 10 mM 3-MA; 100 nM RAPA.Scale: 10 μm.Quantification of g) lipid peroxidation levels and h) LD content in H9c2 cells using flow cytometry.i) After induction with 5 μM Erastin for 20 h, the LD content in norma

Figure 7 .
Figure 7. Evaluation of the correlation between ferroptosis and MIRI.a) TTC staining of heart slices.b, c) Scanned and magnified images of b) H&E stained and c) Masson's trichrome stained heart slices obtained from mice subjected to sham surgery or 30 min/4 weeks of I/R induction.Scale: 1000 μm (left), 100 μm (right).d-g) Measurement of d) AST, e) CK, f) CK-MB, and g) LDH1 levels in mice; n = 6.h, i) CLSM images showing sections of heart tissue labeled with h) C11-BODIPY 581/591 and i) GPX4.Scale: 50 μm.j, k) Representative images and quantification of the nonheme iron-positive area in heart sections stained with Prussian blue (enhanced with DAB) (n = 5).Scale bar: 50 μm.l-n) Serum l) GPX4 levels, m) 4-HNE levels, and n) MDA levels in mice; n = 6.o) The mouse myocardium, particularly the ultrastructure of the anterior wall of the left ventricle, was observed by TEM.(Black arrows: autophagic vacuole; *: LD.) Scale: 2 μm.Most of these groups, not groups b or c, underwent 30 min/24 h I/R and sham surgery.The statistical significance of the differences in the results presented in d-g and k-n was assessed by Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.Different letters indicate significant differences between labeled groups (p < 0.05).

Figure 8 .
Figure 8. Monitoring the changes of LDs and GPX4 in MIRI mice.a) Schematic illustration of the imaging of the I/R injury hearts at different times.b) Representative ex vivo fluorescence image of hearts and sections in different groups (Sham, I/R-6, I/R-9, I/R-12, and I/R-24 h).c) Statistical analysis showing the average radiation efficiency in isolated hearts in the Sham, 6, 9, 12, and 24 h reperfusion groups; n = 5. d-f) Observation of mouse heart sections by different methods.The images are from the sham, 9, and 24 h reperfusion groups.d) CLSM images were used to measure the LD content in heart sections.Scale: 50 μm.e) CLSM images were used to measure the activity of GPX4 in heart sections.Scale: 50 μm.f) Oil red O staining revealed the LD distribution in heart sections.Scale: 50 μm.The statistical significance of differences in c was determined by Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001.Different letters indicate significant differences between the labeled groups (p < 0.05).

Figure 9 .
Figure 9. Validation of the therapeutic function of CQ. a) Schematic showing the evaluation of mice with MIRI after treatment with CQ. b) Scanned and magnified images of Masson's trichrome staining of heart slices taken from control mice or mice subjected to 30 min/4 weeks I/R injury and injected with saline or CQ.c) Analyses of heart sections stained with Prussian blue (enhanced with DAB) from the control (untreated), I/R, and CQ treatment group mice.d) Measurement and comparison of the nonheme-iron-positive area in control, I/R and CQ treatment group mice; n = 5. e) Measurement of serum 4-HNE levels in control, I/R, and CQ treatment group mice; n = 6.f-i) Observation of mouse heart section images obtained via different methods.The images are from control, I/R, and CQ treatment group mice.f) Confocal images were used to observe LDs in heart sections labeled with TPABTBP.Scale: 50 μm.g) Fluorescence images were used to observe the activity of GPX4 in heart sections.Scale: 50 μm.h) Corresponding images of heart slices stained with Oil red O. Scale: 50 μm.i) The myocardium was observed by TEM to determine the ultrastructure of the anterior wall in the left ventricle.(Black arrows: autophagic vacuole.)Scale: 2 μm.j-m) Measurements of serum j) AST levels, k) CK levels, l) CK-MB levels, and m) LDH1 levels in control, I/R, and CQ treatment group mice; n = 6.The statistical significance of differences shown in j-m was determined by Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001.Different letters indicate significant differences between the labeled groups (p < 0.05).